Studies of Enzyme-Ligand Complexes Using Dynamic Fluorescence Anisotropy. I. The Substrate-Binding Site of Malate Dehydrogenase
- 1 January 1986
- journal article
- Published by Walter de Gruyter GmbH in Biological Chemistry Hoppe-Seyler
- Vol. 367 (1) , 95-102
- https://doi.org/10.1515/bchm3.1986.367.1.95
Abstract
The substrate binding site of pig mitochondrial malate dehydrogenase was characterized using complexes of the enzyme with the substrate analogue 8-hydroxypyrene-1,3,6-trisulfonate with and without the addition of coenzymes. The rotational mobility of the fluorescent dye within the binding site was examined with the aid of a multi-frequency phase-fluorimeter. Together with absorption, circular dichroism and fluorescence spectroscopy, conformational changes of the substrate binding site could be defined. The dye was generally found to be immobilized in the binding site. Addition of NADH to the binary complex caused strengthening of a hydrogen bond and further loss of mobility, whereas NAD enlarged the space available for motion of the dye with concomitant loss of the hydrogen bridge.Keywords
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